Department of Biosciences, University of Exeter, Exeter EX4 4PE, United Kingdom.
Proc Natl Acad Sci U S A. 2011 Mar 1;108(9):3618-23. doi: 10.1073/pnas.1015839108. Epub 2011 Feb 11.
In many cell types, bidirectional long-range endosome transport is mediated by the opposing motor proteins dynein and kinesin-3. Here we use a fungal model system to investigate how both motors cooperate in early endosome (EE) motility. It was previously reported that Kin3, a member of the kinesin-3 family, and cytoplasmic dynein mediate bidirectional motility of EEs in the fungus Ustilago maydis. We fused the green fluorescent protein to the endogenous dynein heavy chain and the kin3 gene and visualized both motors and their cargo in the living cells. Whereas kinesin-3 was found on anterograde and retrograde EEs, dynein motors localize only to retrograde organelles. Live cell imaging shows that binding of retrograde moving dynein to anterograde moving endosomes changes the transport direction of the organelles. When dynein is leaving the EEs, the organelles switch back to anterograde kinesin-3-based motility. Quantitative photobleaching and comparison with nuclear pores as an internal calibration standard show that single dynein motors and four to five kinesin-3 motors bind to the organelles. These data suggest that dynein controls kinesin-3 activity on the EEs and thereby determines the long-range motility behavior of the organelles.
在许多细胞类型中，双向长程内体运输是由相反的马达蛋白动力蛋白和驱动蛋白-3 介导的。在这里，我们使用真菌模型系统来研究这两种马达蛋白如何在早期内体（EE）运动中协同作用。先前的研究报道称，驱动蛋白-3 家族的成员 Kin3 和细胞质动力蛋白介导了真菌 Ustilago maydis 中 EE 的双向运动。我们将绿色荧光蛋白与内源性动力蛋白重链和 kin3 基因融合，并在活细胞中可视化这两种马达蛋白及其货物。虽然驱动蛋白-3 被发现存在于顺行和逆行 EE 上，但动力蛋白马达仅定位于逆行细胞器。活细胞成像显示，逆行运动的动力蛋白与顺行运动的内体结合会改变细胞器的运输方向。当动力蛋白离开 EE 时，细胞器又切换回基于顺行驱动蛋白-3 的运动。定量光漂白并与核孔作为内部校准标准进行比较表明，单个动力蛋白马达和四到五个驱动蛋白-3 马达结合到细胞器上。这些数据表明，动力蛋白控制 EE 上驱动蛋白-3 的活性，从而决定了细胞器的长程运动行为。
